Analysis of class I phosphoinositide 3-kinase autophosphorylation sites by mass spectrometry |
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Authors: | Czupalla Cornelia Nürnberg Bernd Krause Eberhard |
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Affiliation: | Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universit?t, Universit?tsstr 1, Geb?ude 22.03, 40225 Düsseldorf, Germany. |
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Abstract: | This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms beta and gamma by mass spectrometry. Since discrimination and suppression effects prevented the immediate detection and sequencing of phosphopeptides in complex mixtures, a strategy was applied that involved (32)P-radiolabeling of the phosphoproteins, cleavage of the phosphoproteins with several proteases and/or cyanogen bromide, separation of the resulting peptide mixtures by micro-reversed-phase liquid chromatography, and mass spectrometric analysis of fractions containing phosphopeptides. As a result the primary autophosphorylation sites of phosphoinositide 3-kinase p110beta and p110gamma subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively. |
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